Pre-Conceptional Exposure to Glyphosate Affects the Maternal Hepatic and Ovarian Proteome

Abstract Exposure to glyphosate (GLY), a commonly used herbicide, is supported by urinary detection and associated with shortened gestation in women. This study tested the hypothesis that chronic low-dose pre-conceptional GLY exposure would affect maternal ovarian function mid- and post-gestation. Mice (C57BL/6; n = 40) were exposed per os to saline vehicle control (CT; n = 20) or GLY (2 mg/kg; n = 20) daily for 10 weeks starting at 7 weeks of age. Post-exposure, females were impregnated and euthanized at gestation day 14 (GD14) or post-weaning (PW). Pregnancy success was reduced from 75% to 55% by GLY exposure. No treatment effect (p > .05) on body weight, maternal serum 17β-estradiol, or litter size was noted. Ovarian weight was unaffected or reduced (p < .05) by GLY in GD14 and PW dams, respectively. Exposure to GLY decreased (p < .05) PW ovarian secondary follicle number with no other follicle composition impacts. Protein abundance analysis by LC-MS/MS identified that GLY altered (p < .05) 26 ovarian and 41 hepatic proteins in GD14 dams and 39 hepatic proteins in PW dams. In GD14 dams, GLY increased ovarian protein abundance of SEC16A (p < .05; 29-fold) and hepatic RPS27L and GM4952 (p < .05; ∼4-fold). In both GD14 and PW dams, GLY exposure increased (p < .05) hepatic RPS4 and decreased (p < .05) ECHDC3. Pathway analysis using DAVID identified 10 GLY hepatic pathway targets with FDR ≤ 0.07 in GD14 dams.

Ovotoxicant exposures compromise the ovary in a variety of ways including but not limited to, depletion of primordial germ cells (Borman et al., 2000;Hoyer and Keating, 2014;Mattison and Schulman, 1980), primordial follicle growth hyperactivation (Keating et al., 2011), induction of DNA damage (Ganesan et al., 2013), and alteration to follicular growth and viability (Hoyer, 2001) at each stage of folliculogenesis. In addition, ovotoxicants can affect steroidogenesis and result in premature follicular loss (Krarup, 1969) and early menopause as a consequence (Hoyer, 2001;Hoyer and Keating, 2014). Additionally, negative impacts on pre-and peri-pubertal sexual maturation in exposed animals are documented (Hatch et al., 2011;€ Ozen and Darcan, 2011;Yang et al., 2016). Further, ovotoxicant exposure during fetal development can harm offspring germ cell number and quality (Brieño-Enr ıquez et al., 2015;Johansson et al., 2016). Glyphosate (GLY) is a widely used herbicide in urban and rural environments (Gillezeau et al., 2019). Since the mid-1990s, the introduction of GLY-resistant crops resulted in a dramatic increase in GLY agricultural usage for weed control (Benbrook, 2016). Commercially, GLY salts are mixed with surfactants to increase penetrative capacity in plants (Benbrook, 2016) and referred to as GLY-based herbicides (GBHs). Both GLY and GBHs are also broadly utilized for weed control in home gardens, railways, and roadways.
The presence of GLY residues in food stuffs (Bøhn et al., 2014;Granby et al., 2003;Zoller et al., 2018Zoller et al., , 2020 and in human urine (Lemke et al., 2021;Mills et al., 2017;Soukup et al., 2020) supports human GLY exposure. A review of human GLY studies from Europe and the United States concluded lack of GLY health risk based upon the level of exposure being lower than the proposed acceptable daily intake established by the European Food Safety Authority (0.5 mg/kg/body weight) (Niemann et al., 2015). The level of GLY and/or the main metabolite aminomethylphosphonic acid (AMPA) concentrations in human urine samples relative to known ingested concentrations determined that approximately 1%-6% of ingested GLY was recoverable in urine (Faniband et al., 2021), whereas GLY is readily excreted through feces and urine (Williams et al., 2000).
In rodents, GLY exposure increased atretic ovarian follicle number and decreased antral follicle number at doses of 126 or 315 mg/kg/day (Hamdaoui et al., 2018). Increased granulosa and theca cell proliferation and decreased follicle-stimulating hormone receptor and growth and differentiation factor 9 mRNA level at a GLY dose of 2 mg/kg in lambs has been observed (Alarc on et al., 2019). In contrast, no observable effects of 5 or 10 weeks of 2 mg/kg GLY exposure on follicle number or other ovarian endpoints have been reported in mice . Further, a GLY dose response study determined no effect of 20 weeks of GLY (0.25-1.5 mg/kg) exposure on follicle number with the exception of the upper 2 mg/kg dose, in which increased follicle number relative to control treated mice was noted, in the absence of an effect on estrous cyclicity in mice . Exposure to 500 mM GLY impaired oocyte maturation through reduced germinal vesicle breakdown proposed to be due to increased reactive oxygen species generation . Interestingly, exposure to 0.25-2 mg/kg GLY altered ovarian proteins with mitochondrial and oxidative stress response functions . The F2 offspring of F1 rats exposed both perinatally and during lactation to GBH (2 or 200 mg/kg) had delayed fetal growth and congenital anomalies at the higher GBH dose (Milesi et al., 2018). In humans, GLY urinary concentration of pregnant women was correlated with shortened gestation length (Lesseur et al., 2022;Parvez et al., 2018). Thus, there is rationale for consideration of GLY as a reproductive toxicant.
Based upon evidence of GLY-induced alterations to ovarian and reproductive endpoints and the recognized ability of toxicant exposures to have multi-and trans-generational effects (Milesi et al., 2021;Patisaul, 2021), this study investigated the hypothesis that GLY exposure would affect ovarian endpoints in dams due to pre-conceptional exposure. An exposure level of 2 mg/kg/day was chosen, based upon lack of observable effects of this dose on reproductive and developmental endpoints by the Environmental Protection Agency (ATSDR, 2020). An exposure duration of 70 days was conducted to encompass an entire round of folliculogenesis (Hoyer, 2001) to evaluate if GLY exposure affected ovarian follicular composition and the maternal liver and ovary proteome.
Animal dosing and breeding. All animal experiments were approved by the Institutional Animal Care and Use Committee at Iowa State University. Female C57Bl/6 mice were purchased from Jackson Laboratories aged 6 weeks. After 1 week of acclimation, mice were randomly split into 2 groups: one group received saline per os from a pipette tip as vehicle control (CT; n ¼ 20) and the other group received GLY (2 mg/kg; n ¼ 20) diluted in saline once per day for 10 weeks by the same route. Mice were maintained under controlled room temperature (21 C-22 C) and 12 h light: dark cycles with ad libitum access to food and water.
Food intake and body weight were measured weekly. Food consumption was calculated as the mean chow (g) eaten per cage per week.
After 10 weeks of CT or GLY exposure, females were housed with C57Bl/6 males for approximately 5 days and the appearance of a vaginal plug was monitored daily. The monitoring of the vaginal plug observance was designated as gestational day 1 (GD1). The pregnancy success for each group was calculated as: the number of pregnant dams as a percent of the total number of females housed with males. At GD14, CT and GLY females (n ¼ 10 per treatment) were euthanized for tissue collection and embryo enumeration and designated as GD14. The remainder of the pregnant dams (n ¼ 10 per treatment) completed pregnancy and lactation and were euthanized 1-week post-weaning and designated as PW. All animals in the study were euthanized by CO 2 asphyxiation followed by cervical dislocation.
Tissue collection. Liver, spleen, and ovaries were collected from GD14 and PW dams, weighed and the liver and ovary were immediately either snap frozen in liquid nitrogen or fixed in 4% PFA overnight at 4 C for histological analysis. One ovary was frozen and stored at À80 C for protein analysis, and another was fixed in 4% PFA for hematoxylin and eosin staining. Blood was collected by cardiac puncture, stored on ice and centrifuged for 15 min at 10 000 rpm for serum separation.
Serum 17b-estradiol and progesterone hormone level quantification. The level of 17b-estradiol and progesterone in serum was quantified by the Ligand Assay & Analysis Core of the Center for Research in Reproduction, University of Virginia with 2 technical replicates per sample.
Histological analysis. Ovaries were fixed in 4% PFA for 24 h, transferred to 70% ethanol, and then paraffin embedded. One ovary from each mouse was sectioned (5 lM thickness) and every 6th section was mounted and stained with hematoxylin and eosin. Oocyte-containing follicles and corpora lutea (CLs) were identified and counted in every 12th section. Healthy follicles were classified and counted as described previously (Flaws et al., 1994). Atretic follicles were differentiated from healthy follicles by the appearance of pyknotic bodies and intense eosinophilic staining of oocytes.
LC-MS/MS analysis. Ovarian and liver tissue homogenates were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Briefly, protein extracts were reduced with dithiothreitol, with the cysteine groups modified with iodoacetamide, and digested overnight with trypsin/Lys-C. The addition of formic acid stopped digestion before drying down samples in a SpeedVac. Samples were desalted using C18 MicroSpin Columns (Nest Group SEM SS18V) before drying again in a SpeedVac. Peptide retention time calibration (PRTC) standard (Pierce part no. 88320) was spiked into the sample to serve as an internal control. The peptides were then separated by liquid chromatography and analyzed by MS/MS by fragmenting each peptide using a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The resulting intact and fragmentation pattern was compared with a theoretical fragmentation pattern (from either MASCOT or Sequest HT) for protein identification. Label-free quantification technique used the Minora Feature Detector to detect and quantify isotopic clusters. The PRTC areas were used to normalize the data between samples. Proteins were identified by searching databases with both MS and MS/MS data using Mascot software as described previously (Xia and Wishart, 2016).
Statistical analysis. Statistical analysis was conducted by unpaired t test using Prism 9.0.1 software (GraphPad Prism). Statistical significance was defined as p < .05. For the pathway analyses based on LC-MS/MS results, proteins that differed from CT at p < .1 were included. Biological pathways altered by GLY exposure were predicted using DAVID v 6.8 software. The number for "Gene Found" represented the number of genes identified from proteomics analyses. The number for "Gene Pathway" represented the total number of genes in the particular pathway as found in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The false discovery rate (FDR) for every presented pathway was also calculated using DAVID v 6.8 software. The results in barcharts are represented mean 6 standard error of mean (SEM).
Samples identification methods. The sample identity for follicle counting, 17b-estradiol, progesterone, and proteomic analyses was not known to the investigator conducting the analyses.

Influence of GLY Pre-Conceptional Exposure on Body Weight
The mean body weight for females at the end of 10 weeks of dosing before mating with males did not differ for GLY-exposed females (p > .05; CT ¼ 20.25 6 0.3 g; GLY ¼ 21 6 0.4 g). The food eaten per cage per week during the dosing period also did not differ between CT and GLY groups. Pre-conceptional GLY exposure did not affect body weight at GD14 (p > .05; CT ¼ 29.07 6 1.1 g; GLY ¼ 31.07 6 0.5 g; Figure 1A) or PW (p > .05; CT ¼ 24.30 6 0.5 g, GLY ¼ 23.73 6 1.1 g; Figure 1B).

Effect of Pre-Conceptional GLY Exposure on Pregnancy Success
Pregnancy success in CT mice was 75% with 15 out of 20 females conceiving and delivering offspring (data not shown). In GLYexposed mice, pregnancy success was reduced to 55% with 11 of 20 females delivering offspring (data not shown).

Impact of Pre-Conceptional GLY Exposure on Circulating Serum 17b-Estradiol and Progesterone Levels
The levels of 17b-estradiol and progesterone were measured only in the serum obtained from the GD14 dams due to a low volume of serum obtained from PW dams. There was no treatment effect on serum 17b-estradiol (p < .05; CT ¼ 12.14 6 3.2 pg/ ml; GLY ¼ 10.61 6 1.4 pg/ml; Figure 4A) or progesterone (p < .05; CT ¼ 12.7 6 3.2 ng/ml; GLY ¼ 15.5 6 2.5 ng/ml; Figure 4B) at GD14.

Effect of Pre-Conceptional GLY Exposure on Litter Size
The mean number of embryos per litter was similar for GD14 GLY exposed compared with CT dams (p > .05; CT ¼ 8.6 6 0.5; GLY ¼ 7.3 6 0.7; Figure 5A). The mean number of pups born alive per litter did not differ between CT and GLY exposed dams (p > .05; CT ¼ 7.3 6 0.6; GLY ¼ 5.8 6 0.9; Figure 5B).
Effect of Pre-Conceptional GLY Exposure on GD14 Ovarian and Hepatic Proteome In GD14 mice, GLY exposure altered (p < .05) the abundance of 26 ovarian proteins, relative to CT mice (Table 1) with an FDR > 0.05. Out of those, the abundance of 2 proteins was increased and 4 proteins was decreased with a fold change > 2 in GLY compared with CT dams ( Figure 8A). Protein transport protein sec16 (SEC16A) was increased 29-fold by GLY exposure. Pathway analyses of the ovarian altered proteins performed with DAVID did not identify possible targets with FDR < 0.1.
Impact of Pre-Conceptional GLY Exposure on the Hepatic Proteome of PW Dams GLY-exposed PW dams had alterations (p < .05) to the abundance of 39 hepatic proteins relative to CT mice (Table 4; FDR > 0.05), including the xenobiotic biotransformation proteins cytochrome P450 isoform 2D22 (CYP2D22; 0.8-fold decrease) and dimethylaniline monooxygenase 1 (FMO1; 0.9-fold decrease). The abundance of 4 proteins were increased and 3 proteins were decreased by GLY exposure with a fold change > 2 (Figure 9). Pathway analyses of the hepatic proteome performed with DAVID platform did not identify targets with FDR < 0.1.

DISCUSSION
The potential risks of GLY chronic pre-conceptional exposure on ovarian follicle composition and the proteome of the ovary and liver both during and after pregnancy was investigated in this study. GLY's widespread agricultural usage became possible due to development of GLY-resistant crops in the early 1990s (Benbrook, 2016). By 2014, approximately 240 million pounds were sprayed annually on U.S. agricultural fields (Benbrook, 2016). Excretion of GLY and its main metabolite, AMPA, have been detected in human urine (Connolly et al., 2018;Curwin et al., 2007;Faniband et al., 2021;Knudsen et al., 2017). In commercial formulations, GLY is mixed with surfactants to increase its penetrative qualities to inhibit weed growth (Mesnage et al., 2019).  Female C57Bl/6 mice were exposed to saline vehicle control (CT) or GLY (2 mg/ kg) pre-conceptionally for 10 weeks daily per os. (A) Liver and (B) spleen weight in GD14 dams (n ¼ 7 for both CT and GLY). (C) Liver and (D) spleen weight in PW dams (n ¼ 8 for CT, n ¼ 3 for GLY). Data points represent mean 6 SEM. Figure 1. Impact of pre-conceptional GLY exposure on GD14 and PW female body weight. Female C57Bl/6 mice were exposed to saline vehicle control (CT) or GLY (2 mg/kg) pre-conceptionally for 10 weeks daily per os. A, GD14 dam weight (n ¼ 7 for both CT and GLY). B, PW dam body weight (n ¼ 8 for CT, n ¼ 3 for GLY). Data points represent mean 6 SEM. Figure 3. Impact of pre-conceptional GLY exposure on GD14 and PW ovarian weight. Female C57Bl/6 mice were exposed to saline vehicle control (CT) or GLY (2 mg/kg) pre-conceptionally for 10 weeks daily per os. Ovarian weight of (A) GD14 (n ¼ 7 for both CT and GLY) and (B) PW dams (n ¼ 8 CT, n ¼ 3 for GLY). Data points represent mean 6 SEM. Asterisk represents difference from CT; p < .05. Figure 5. Effect of pre-conceptional GLY exposure on litter size. Female C57Bl/6 mice were exposed to saline vehicle control (CT) or GLY (2 mg/kg) pre-conceptionally for 10 weeks daily per os. A, The mean number of embryos per litter at GD14 (n ¼ 7 for both CT and GLY). B, The mean number of pups born alive per litter (n ¼ 8 for CT, n ¼ 4 for GLY). N represents the litter number. Data points represent mean 6 SEM. . Impact of pre-conceptional GLY exposure on circulating 17b-estradiol and progesterone in GD14 dams. Female C57Bl/6 mice were exposed to saline vehicle control (CT) or GLY (2 mg/kg) pre-conceptionally for 10 weeks daily per os. (A) 17b-estradiol (n ¼ 7 for both CT and GLY) and (B) progesterone level (n ¼ 3 for CT, n ¼ 5 for GLY). Data points represent mean 6 SEM. However, the same dose impacted the ovarian proteome after a longer exposure paradigm of 20 weeks with particular effects on mitochondrial and oxidative stress proteins. Additionally, a higher number of ovarian follicles present within the ovary were noted implicating the potential of GLY to affect ovarian function . Whether these impacts resulted in fertility consequences remain unclear.
In this study, the effects of a chronic pre-conceptional exposure of GLY on maternal ovarian follicle number and the ovarian and hepatic proteome were investigated. The administration route chosen mimicked both oral and chronic exposure. The dose was chosen based upon previous studies by our group and others (Alarc on et al., 2019;  and the duration of 70 days encompassed an entire round of folliculogenesis (Butcher et al., 1974;Rajkovic et al., 2006).
Though previous studies report a negative impact of GLY exposure on body weight (Ait Bali et al., 2017;Jasper et al., 2012;Yousef et al., 1995), a difference in the weight gain between CT and GLY exposed mice after 10 weeks of exposure was not demonstrated. A similar lack of an effect of 2 mg/kg GLY on female body weight after 5 or 10 weeks of exposure was also shown in our previous studies . Additionally, there was no impact of GLY exposure on food intake, with the caveat that the food intake measurements were per cage rather than total caloric intake per individual mouse. Thus, no evidence of an impact of glyphosate exposure on food consumption or weight gain was observed.
A reduction in pregnancy was noted due to GLY exposure. The mice were approximately 17 weeks of age at the time of breeding, thus, this age could influence pregnancy outcomes independent of chemical exposure. Although difficult to conclude if pregnancy loss was increased from this study design, a negative impact on uterine morphology and decidualization was reported previously in female rats that were exposed to 2 mg/kg of GBH neonatally (Ingaramo et al., 2016), potentially affecting pregnancy success. An association between urinary GLY level and shortened gestation (Lesseur et al., 2022;Parvez et al., 2018) and oxidative stress (Eaton et al., 2022) has also been reported in women. Previous studies reported a GLY impact on maternal weight (Ren et al., 2018;Teleken et al., 2020), however this was not noted in the current study in either the mid-gestation or post-partum mice. There was also no impact of GLY on liver or spleen weight. Interestingly, ovarian weight was reduced in PW mice due to preconceptional GLY exposure but this effect was not observed in Figure 7. Effect of pre-conceptional GLY exposure on ovarian follicle number in PW dams. Female C57Bl/6 mice were exposed to saline vehicle control (CT) or GLY (2 mg/kg) pre-conceptionally for 10 weeks daily per os.  dams. Female C57Bl/6 mice were exposed to saline vehicle control (CT) or GLY (2 mg/kg) pre-conceptionally for 10 weeks daily per os. A, The number of CL in the ovaries from GD14 dams (n ¼ 5 for both CT and GLY). B, The number of atretic follicles in ovaries of GD14 dams (n ¼ 5 for both CT and GLY). N represents the number of dams. Data points represent mean 6 SEM. GD14 mice. These findings could indicate an effect of GLY on ovarian composition intensified by ovarian aging and longer term studies are warranted.
The ovarian and placental hormones progesterone and 17bestradiol are important for maintaining pregnancy, and dysregulation of their levels could result in a spectrum of pregnancy complications including miscarriage (Berkane et al., 2017;Kumar and Magon, 2012). Several previous studies demonstrated the potential of GBHs to act as endocrine disruptors impacting the circulating levels of the progesterone and/or 17bestradiol (Perego et al., 2017;Ren et al., 2018;Wrobel, 2018). However, previous studies from our group did not observe changes in estrous cyclicity or in circulating levels of 17b-estradiol or progesterone in female mice after 10 and/or 20 weeks of 2 mg/kg of GLY exposure ). In the current study, circulating levels of 17b-estradiol and progesterone were similar between treatments in pregnant dams in contrast with studies documenting 17b-estradiol and progesterone effects during pregnancy when exposed to higher concentrations of GBHs (Ren et al., 2018). Thus, pre-conceptional GLY exposure did not result in observable changes to 17b-estradiol and progesterone during gestation in this paradigm.
Our previous studies on the impact of GLY exposure at 2 mg/ kg for 5 or 10 weeks on ovarian follicle number did not reveal .024608 Elongation factor 1-alpha 2 À1.5525 .021982 Protease, serine 1 (trypsin 1) À1.7669 .021208 Figure 8. Impact of pre-conceptional GLY exposure on ovarian and hepatic proteome of GD14 dams. Female C57Bl/6 mice were exposed to saline vehicle control (CT) or GLY (2 mg/kg) pre-conceptionally for 10 weeks daily per os. (A) Ovary (n ¼ 5 for CT, and n ¼ 4 for GLY) and (B) liver (n ¼ 5 for CT, and n ¼ 4 for GLY) were collected at GD14 and proteins analyzed by LC-MS/MS. Bioinformatic analyses were performed to determine differences in protein abundance due to GLY exposure relative to CT. The pink dots in the volcano plots above the horizontal dotted line indicate proteins that differed in abundance with fold change > 2 and p < .05.
changes in the number of follicles at any stage of development compared with control treated females . However, after 20 weeks of the identical chronic GLY exposure as in the current study, females had a greater number of ovarian primordial and antral follicles compared with control mice . We did not observe a difference in the number of CL or atretic follicles in pre-conceptional GLYexposed GD14 compared with CT mice. Contradictory to our findings, mice exposed to GLY dissolved in drinking water (0.5%) had higher numbers of atretic follicles (Ren et al., 2018). In Table 3. KEGG pathways altered in liver of GLY-exposed GD14 dams  PW dams, decreased secondary follicle number was observed due to GLY exposure, which may be explanatory for the reduced ovarian weight observed. These divergent GLY effects are likely explained by difference in dosing paradigms between studies.
To identify modes of action of pre-conceptional GLY exposure on the ovary and liver, a non-targeted approach was taken and whole ovarian or hepatic protein was analyzed by LC-MS/ MS. The impact of GLY on the maternal GD14 ovary included changes to 26 proteins with the abundance of SEC16A being increased by 29-fold. SEC16A is critical in its regulatory role of several proteins' endoplasmic reticulum conventional and unconventional secretion (Piao et al., 2017;Supek et al., 2002;Watson et al., 2006), and is involved in nutrient-induced autophagy (Zahoor and Farhan, 2018). Such a dramatic increase due to GLY exposure in pregnancy could be a sign of dysregulation of metabolic processes. The level of ADP-ribose glycohydrolase MACROD1, that participates in post-translational modifications of target proteins, was increased 1.6-fold in the ovaries of GD14 dams exposed to GLY. This protein is activated in response to environmental triggers (Feijs et al., 2013) and involved in DNA damage response mechanisms (Yang et al., 2020). Expressed in many tissues, MACROD1 abundance is elevated in several types of cancers (Feijs et al., 2020). In addition, among the proteins demonstrating increased abundance in the ovarian tissues from the pregnant dams exposed to GLY, heat shock 70 kDa protein 4 (HSPA4) was increased (1.1-fold). Increased activity of HSPA4 and other heat shock proteins of the HSP110 family was implicated in carcinogenesis and drug resistance (Gotoh et al., 2004;Kimura et al., 2016). The increase in abundance of these proteins in the ovaries from GLY-exposed dams could indicate that, even at low concentrations, ovarian molecular changes are observed due to pre-conceptional GLY exposure. It is important to note that these findings represent a snapshot of the ovarian impacts and whether these changes could influence gestational outcomes or long-term fertility cannot be concluded.
There were changes to the abundance of 41 and 39 hepatic proteins in the GD14 and PW livers, respectively. The liver, as an important metabolic organ, serves as the major site for xenobiotic biotransformation with dramatically higher levels of Phase I and Phase II biotransformation enzymes than other organs (Gu and Manautou, 2012). This study identified a preconceptional GLY impact on the level of maternal hepatic xenobiotic Phase I biotransformation enzymes including CYP2C40, CYP2C37, and CYP2D10, and the Phase II biotransformation proteins SULT1A1 and GSTA5. In the livers of PW mice, two Phase I biotransformation enzymes, CYP2D22 and FMO1, were decreased due to GLY exposure. Interestingly, in both GD14 and PW dams, hepatic 40S ribosomal protein S4 (RPS4) level was increased and Enoyl-CoA hydratase domain-containing protein 3, mitochondrial (ECHDC3) abundance was decreased. This similar pattern of RPS4 and ECHDC3 proteins at both GD14 and PW may indicate a persistent influence of GLY exposure. Hepatic proteomic pathway analyses demonstrated that pre-conceptional GLY exposure changed proteins that represented specific pathways during pregnancy. Among the potential pathway targeted by GLY at GD14 with FDR < 0.07 were chemical carcinogenesis, steroid hormone biosynthesis, retinol metabolism, and metabolic pathways, all of which are important for maintaining pregnancy.
Taken together, lack of observed impacts of GLY exposure on body weight, liver or spleen weight, food intake, GD14 ovary weight, GD14 ovarian follicle composition, GD14 17b-estradiol and progesterone levels suggest an absence of endocrine disruption and overt toxicity. Reduced PW secondary follicle number and ovarian weight and an altered ovarian and hepatic proteome suggest that pre-conceptional chronic GLY exposure may affect maternal ovarian and hepatic function. Further studies to evaluate any long term impacts of the changes observed herein are needed.